The cytochrome P-450 dependent monooxygenase system plays a pivotal role in both the detoxification and bioactivation of drugs, environmental contaminants, and other potential chemical toxicants. The balance between detoxification and activation is largely dependent on the relative amounts and activities of different isozymes of cytochrome P-450. The long-term objective of the research proposed in this application is to design irreversible inhibitors of specific isozymes of cytochrome P-450. Such isozyme-specific inhibitors could be used in vivo either 1) diagnostically to assess the role of the various cytochromes in mediating or protecting against chemical toxicity or 2) therapeutically to redirect the metabolism of xenobiotics from potentially harmful to innocuous pathways. This proposal will focus on the mechanism, isozyme specificity, and structural requirements of the suicide inactivation of rat liver cytochrome P-450 by chloramphenicol. As the only known suicide substrate of cytochrome P-450 which acts by virtue of the modification of the protein rather than the heme moiety, chloramphenicol or one of its analogs should prove a unique tool for studying and modulating the various functions of the enzyme. Emphasis will first be placed on elucidation of the mechanism by which a single isozyme of rat liver cytochrome P-450 is inactivated in vivo upon covalent modification of specific amino acid residues in the protein by metabolites of chloramphenicol. Subsequent studies will focus on determining the specificity of chloramphenicol as an inhibitor of different isozymes of rat liver cytochrome P-450 and on elucidating which structural features of the chloramphenicol molecule are responsible for its effectiveness and specificity as a suicide substrate of cytochrome P-450. These studies should provide the rational basis for the design of isozyme-specific inhibitors for modulating monooxygenase function in vivo.
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